Hemes are among the most prevalent and versatile cofactors in biological systems. One of the factors affecting the adaptability of these cofactors to a wide variety of different biological needs is the ease with which the redox state, and within each redox state, the electronic state of the iron may be adjusted by porphyrin substituents and by protein environment. The protein not only provides axial ligands, but may severely restrict orientation of the ligands relative to each other and the heme macrocycle. Among possible spin states, low-spin Fe(II) and (III) are present in most cytochromes a, b, c, d, and f. A hallmark of the electronic ground state of all Fe(III) hemes is their para-magnetism; hence NMR, EPR, Mossbauer, and MCD spectroscopies are rich in information about the molecular and electronic structure of the metal center. During the past ten years, with the use of appropriate model compounds and spectroscopic tools, we have sought to learn if axial ligand plane orientation is an important function of the protein environment, and more recently, to learn what factors determine the electronic ground state of the iron. During the next grant period we will concentrate on three projects: I. The effect(s) of axial ligands, heme substituents, and heme ring reduction state on the electronic ground state of low-spin Fe(III) porphyrins and reduced ring hemes will be elucidated using a combination of NMR, EPR, Mossbauer, and MCD spectroscopies and x-ray crystallography. II. The importance of axial ligand plane orientation on the reduction potentials and spectroscopic properties of model hemes and heme proteins will be probed using a series of new "cavity-type" iron porphyrins. EPR, NMR, Mossbauer and MCD spectroscopies will be used appropriately to determine the temperatures at which axial ligand rotation ceases, yielding complexes in which the axial ligands are in either parallel or perpendicular planes for both the Fe(III) and Fe(II) states. The solution structures and the dynamics of the Fe(III) complexes will be investigated by 2-D NMR. The temperature dependence of the reduction potentials will be investigated to determine whether, and if so, how the potentials change when ligand rotation ceases. Frozen solutions of diamagnetic Co(III) and Fe(II) porphyrins bound to axial ligands in parallel or perpendicular planes will be investigated by 13C CPMAS (cross polarization magic angle spinning) NMR techniques in order to evaluate their potential for probing axial ligand orientations in model hemes and membrane-bound heme proteins. III. Electronic effects of axial ligands of metalloporphyrins will be probed by investigating the uv photoelectron spectra of a series of porphyrins and their axial ligand complexes in the solid state. Electronic and steric effects of heme substituents and axial ligands will be probed by 57Fe NMR investigations of model hemes bound to two phosphines or one phosphine and one O, N, or S-donor ligand. Electronic effects of axial ligand combinations that are often difficult to differentiate in naturally-occurring systems ((His-His), (His-Met), (His-Lys), (Lys-Lys) and (Met-Met)) will be probed by spectroscopic and redox investigations of a series of six-coordinate model hemes that will be prepared.